This invention is directed to a method for the generation of nucleic acid ligands having specific functions against target molecules using the SELEX process. The invention provides a method of producing candidate mixtures of RNA nucleic acid ligands without using transcription. The instant methods allow SELEX to be performed using modified ribonucleotides that cannot serve as efficient substrates for RNA polymerases.
The dogma for many years was that nucleic acids had primarily an informational role. Through a method known as Systematic Evolution of Ligands by EXponential enrichrnent, termed the SELEX process, it has become clear that nucleic acids have three dimensional structural diversity not unlike proteins. The SELEX process is a method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules and is described in U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990, entitled xe2x80x9cSystematic Evolution of Ligands by EXponential Enrichment,xe2x80x9d now abandoned, U.S. Pat. No. 5,475,096 entitled xe2x80x9cNucleic Acid Ligandsxe2x80x9d, U.S. Pat. No. 5,270,163 (see also WO 91/19813) entitled xe2x80x9cNucleic Acid Ligandsxe2x80x9d each of which is specifically incorporated by reference herein. Each of these applications, collectively referred to herein as the SELEX Patent Applications, describes a fundamentally novel method for making a nucleic acid ligand to any desired target molecule. The SELEX process provides a class of products which are referred to as nucleic acid ligands or aptamers, each having a unique sequence, and which has the property of binding specifically to a desired target compound or molecule. Each SELEX-identified nucleic acid ligand is a specific ligand of a given target compound or molecule. The SELEX process is based on the unique insight that nucleic acids have sufficient capacity for forming a variety of two- and three-dimensional structures and sufficient chemical versatility available within their monomers to act as ligands (form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric. Molecules of any size or composition can serve as targets in the SELEX method. The SELEX method applied to the application of high affinity binding involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific high affinity nucleic acid ligands to the target molecule.
It has been recognized by the present inventors that the SELEX method demonstrates that nucleic acids as chemical compounds can form a wide array of shapes, sizes and configurations, and are capable of a far broader repertoire of binding and other functions than those displayed by nucleic acids in biological systems.
The basic SELEX method has been modified to achieve a number of specific objectives. For example, U.S. patent application Ser. No. 07/960,093, filed Oct. 14, 1992, now abandoned, and U.S. Pat. No. 5,707,796, both entitled xe2x80x9cMethod for Selecting Nucleic Acids on the Basis of Structure,xe2x80x9d describe the use of the SELEX process in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, entitled xe2x80x9cPhotoselection of Nucleic Acid Ligands,xe2x80x9d, now abandoned, U.S. Pat. No. 5,763,177 entitled xe2x80x9cSystematic Evolution of Nucleic Acid Ligands by Exponential Enrichment: Photoselection of Nucleic Acid Ligands and Solution SELEXxe2x80x9d and U.S. patent application Ser. No. 09/093,293, filed Jun. 8 1998, entitled xe2x80x9cSystematic Evolution of Nucleic Acid Ligands by Exponential Enrichment: Photoselection of Nucleic Acid Ligands and Solution SELEX,xe2x80x9d now U.S. Pat. No. 6,001,577, describe a SELEX based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. U.S. Pat. No. 5,580,737 entitled xe2x80x9cHigh-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine,xe2x80x9d describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, which can be non-peptidic, termed Counter-SELEX. U.S. Pat. No. 5,567,588 entitled xe2x80x9cSystematic Evolution of Ligands by EXponential Enrichment: Solution SELEX,xe2x80x9d describes a SELEX-based method which achieves highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule.
The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX process-identified nucleic acid ligands containing modified nucleotides are described in U.S. Pat. No. 5,660,985 entitled xe2x80x9cHigh Affinity Nucleic Acid Ligands Containing Modified Nucleotides,xe2x80x9d that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2xe2x80x2-positions of pyrimidines. U.S. Pat. No. 5,580,737, supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2xe2x80x2-amino (2xe2x80x2-NH2), 2xe2x80x2-fluoro (2xe2x80x2-F), and/or 2xe2x80x2-O-methyl (2xe2x80x2-OMe). U.S. patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled xe2x80x9cNovel Method of Preparation of 2xe2x80x2 Modified Pyrimidine Intramolecular Nucleophilic Displacement,xe2x80x9d describes oligonucleotides containing various 2xe2x80x2-modified pyrimidines.
The SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. Pat. No. 5,637,459 entitled xe2x80x9cSystematic Evolution of Ligands by EXponential Enrichment: Chimeric SELEX,xe2x80x9d and U.S. Pat. No. 5,683,867 entitled xe2x80x9cSystematic Evolution of Ligands by EXponential Enrichment: Blended SELEX,xe2x80x9d respectively. These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules.
The SELEX method further encompasses combining selected nucleic acid ligands with lipophilic compounds or non-immunogenic, high molecular weight compounds in a diagnostic or therapeutic complex as described in U.S. patent application Ser. No. 08/434,465, filed May 4, 1995, now U.S. Pat. No. 6,011,020 Jan. 4, 2000 entitled xe2x80x9cNucleic Acid Complexesxe2x80x9d. Each of the above described patent applications which describe modifications of the basic SELEX procedure are specifically incorporated by reference herein in their entirety.
The central method for identifying nucleic acid ligands to a target is called the SELEX process, an acronym for Systematic Evolution of Ligands by Exponential enrichment and involves (a) contacting the candidate mixture of nucleic acids the target, (b) partitioning between members of said candidate mixture on the basis of affinity to the target, and (c) amplifying the selected molecules to yield a mixture of nucleic acids enriched for nucleic acid sequences with a relatively higher affinity for binding to the target.
In typical embodiments of the SELEX process, the candidate mixture of nucleic acid ligands comprises RNA molecules. Following partitioning step (b) above, the RNA molecules that have higher affinity for the target are reverse transcribed to form a DNA template. This DNA template is then amplified by the Polymerase Chain Reaction (PCR), and the amplified DNA molecules are transcribed in order to provide a new RNA candidate mixture for the next round of the SELEX process.
Although the transcription of DNA templates during the SELEX process to form RNA nucleic acid ligand candidate mixtures is generally efficient, problems can arise when attempting to incorporate modified ribonucleotides into the RNA molecules during transcription. Such modified ribonucleotides increase the functionality and stability of candidate nucleic acid ligands, but are often poor substrates for RNA polymerase. As a result, transcription in the presence of such modified ribonucleotides is often inefficient, leading to poor yields, or does not take place at all. For example, it is often desirable to incorporate 2xe2x80x2-O alkyl ribonucleotides (ribonucleotides that have an alkyl grouping at the 2xe2x80x2 oxygen), such as 2xe2x80x2-OMe (a methyl group at the 2xe2x80x2 oxygen), into the candidate RNA nucleic acid ligands because such ribonucleotides confer great stability to the RNA. However, no transcription takes place in the presence of 2xe2x80x2-OMe ribonucleotides because these modified ribonucleotides are not substrates for RNA polymerase.
Typical SELEX procedures permit the incorporation, at most, of 5 different modified nucleotides into a candidate mixture (one for each of the 4 NTPs incorporated during the elongation phase of transcription, and one nucleoside, NMP or NDP incorporated at the 5xe2x80x2 end at initiation of transcription). With the exception of the 5xe2x80x2 modification, these modifications are distributed randomly, with respect to both number and position, throughout the randomized portion of the transcript. This random distribution can be a disadvantage, particularly when chemically reactive modifications, or modifications which reduce the solubility or stability of the transcript are introduced.
It is an object of the present invention to provide a method for performing the SELEX process in which modified ribonucleotides, such as 2xe2x80x2-OMe ribonucleotides, are efficiently incorporated into RNA candidate mixtures. It is a further object of the invention to provide a method for performing the SELEX process in which modified ribonucleotides can be incorporated at specific positions.
The instant invention provides novel methods for performing the SELEX process to obtain nucleic acid ligands to target compounds. In particular, methods are provided for obtaining candidate mixtures of nucleic acid ligands comprised of RNA without requiring transcription. Instead of transcription, the candidate RNA nucleic acid ligands are prepared by annealing at least partially randomized RNA fragments to at least partially randomized DNA templates. The annealed RNA fragments are then ligated together to form the candidate nucleic acid ligands. The RNA fragments can be fully synthetic, and so can be comprised of modified ribonucleotide subunits that cannot be incorporated into RNA by RNA polymerase during transcription. Thus, the instant invention allows the SELEX process to be performed with more diverse nucleic acid chemistries than was previously possible.